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Working for a better future

REPORT

2-Bromo-3,3,3-trifluoropropene: Skin Irritation to the Rabbit

HLS study number: WAG0010

Version ID: Final

Issue date: 27 September 2012
Huntingdon Life Sciences

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Details of Sponsor and Test Facility

Sponsor American Pacific Corporation

3883 Howard Hughes Parkway

Suite 700

Las Vegas

Nevada

NV 89169

USA

Test facility Huntingdon Life Sciences

Eye Research Centre

Eye

Suffolk

IP23 7PX

UK
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Table of Contents

Details of Sponsor and Test Facility...................................................................................... 2

Compliance with Good Laboratory Practice .......................................................................... 4

Quality Assurance Statement................................................................................................ 5

Contributing Scientist ............................................................................................................ 6

Summary .............................................................................................................................. 7

1. Introduction .................................................................................................................. 8

2. Test substance............................................................................................................. 9

3. Experimental Procedure............................................................................................. 10

3.1 Animal management....................................................................................... 10

3.2 Test substance preparation............................................................................. 10

3.3 Treatment procedure ...................................................................................... 11

3.4 Serial observations ......................................................................................... 11

3.4.1 Clinical signs ....................................................................................... 11

3.4.2 Dermal responses ............................................................................... 11

3.4.3 Interpretation of responses.................................................................. 12

3.4.4 Termination ......................................................................................... 13

3.5 Archives.......................................................................................................... 14

3.6 Deviations from protocol ................................................................................. 14

4. Results....................................................................................................................... 15

4.1 Clinical signs................................................................................................... 15

4.2 Dermal responses........................................................................................... 15

5. Conclusion ................................................................................................................. 16

List of Tables

Table 1 Mean values for erythema and oedema .......................................................17

Table 2 Individual values for erythema and oedema.................................................18

List of Annexes

Annex 1 Weight of evidence ......................................................................................19

Annex 2 GLP Compliance Statement.........................................................................20
Huntingdon Life Sciences 

WAG0010 

Compliance with Good Laboratory Practice 

2-Bromo-3,3,3-trifluoropropene: Skin Irritation to the Rabbit 

The study described in this report was conducted in conlpliance with the following Good 

Laboratory Practice standards and I consider the data generated to be valid. 

The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 

No. 3106, as amended by Statutory Instrunlent 2004 No. 994). 

GECD Principles of Good Laboratory Practice (as revised in 1997), 

ENV/MCICHEM (98) 17. 

EC Commission Directive 2004/10/EC of 11 February 2004 (Official Journal 

No. L 50/44). 

These principles of Good Laboratory Practice are accepted by the regulatory authorities of 

the United States ofAmerica and Japan on the basis of intergovernmental agreements. 

2:::l- ~~&~. :z.p (1.= 

P BRees BSc (Hons) CBiol MSB Date 

Study Director 

Huntingdon Life Sciences 

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Huntingdon Life Sciences 

WAG0010 

Quality Assurance Statement 

2-Bromo-3,3,3-trifluoropropene: Skin Irritation to the Rabbit 

The following inspections and audits have been carried out in relation to this study: 

Study Phase Date(s) of Inspection	 Date of Reporting to Study 

Director and Management 

Protocol Audit	 07 Mar 2012 07 Mar 2012 

Report Audit	 19 Apr 2012-20 Apr 2012 20 Apr 2012 

Process based inspections 

At or about the time this study was in progress il1spections of procedures employed on this 

type of study were carried out. These were conducted and reported to appropriate Company 

Management as indicated below: 

Process Based Inspections Date(s) of Inspection Date of Reporting to 

Management 

Dose administration & 28 Feb 2012 28 Feb 2012 

Observations - Dermal 

Study Management and 13 Jan 2012 16 Jan 2012 

Conduct 

In addition, an inspection of the facility where this study was conducted was carried out on an 

annual basis. These inspections were promptly reported to Company Management. 

'11 ~\-~ 20(L 

T Whatling MRQA	 Date 

Group Manager 

Department of Quality Assurance 

Huntingdon Life Sciences 

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Huntingdon Life Sciences

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Contributing Scientist

2-Bromo-3,3,3-trifluoropropene: Skin Irritation to the Rabbit

Study management

P B Rees BSc (Hons) CBiol MSB

Study Director
Huntingdon Life Sciences

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Summary

A study was performed to assess the skin irritation potential of 2-Bromo-3,3,3-

trifluoropropene to the rabbit. The method followed was that described in:

EEC Methods for the determination of toxicity and other health effects. 

Commission Regulation No. 440/2008. Part B, Method B.4 Acute Toxicity: Dermal 

irritation/corrosion. 30 May 2008.

OECD Guideline for Testing of Chemicals No.404 “Acute Dermal 

Irritation/Corrosion”. Adopted 24 April 2002.

EPA Health Effects Test Guidelines OPPTS 870.2500 Acute Dermal Irritation 

EPA 712-C-98-196. August 1998.

Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration 

of Agricultural Chemicals, Skin Irritation (2-1-4), 12 Nohsan No. 8147, Agricultural 

Production Bureau, November 24, 2000.

Three rabbits received a single four hour, occlusive, dermal administration of approximately 

0.5 mL of the test substance as supplied and were observed for four days.

No dermal reaction was observed in any animal throughout the duration of the study. 

The means of scores for these reactions at approximately 24, 48 and 72 hours after 

administration, calculated separately for each animal, are summarised below:

Means of scores at approximately 24, 48 and 72 hours

Animal number Erythema Oedema

5 0.0 0.0

78 0.0 0.0

79 0.0 0.0

EC trigger values* 2.3 2.3

*Classification Category 2 according to the Globally Harmonised System and 

European Commission Regulation 1272/2008 is triggered if means of scores 

for either effect are  2.3 for two or three animals (or if effects persist to 

Day 14 in at least two animals).

According to the criteria of the Globally Harmonised System and European Commission 

regulation 1272/2008 2-Bromo-3,3,3-trifluoropropene did not require any classification and 

labelling.
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1. Introduction

The study was designed to assess skin irritation potential of 2-Bromo-3,3,3-trifluoropropene

following a single dermal application to rabbits. The test substance may come into contact 

with skin during handling or use.

The study was conducted in compliance with the following guidelines:

EEC Methods for the determination of toxicity and other health effects. 

Commission Regulation No. 440/2008. Part B, Method B.4 Acute Toxicity: Dermal 

irritation/corrosion. 30 May 2008.

OECD Guideline for Testing of Chemicals No.404 “Acute Dermal 

Irritation/Corrosion”. Adopted 24 April 2002.

EPA Health Effects Test Guidelines OPPTS 870.2500 Acute Dermal Irritation 

EPA 712-C-98-196. August 1998.

Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration 

of Agricultural Chemicals, Skin Irritation (2-1-4), 12 Nohsan No. 8147, Agricultural 

Production Bureau, November 24, 2000.

The albino rabbit was chosen as it has been shown to be a suitable model for skin irritation 

studies and is the animal recommended in the test guidelines.

The amount of test substance administered was chosen in compliance with the guidelines.

The protocol was approved by the Study Director and Huntingdon Life Sciences 

Management on 6 March 2012 and by the Sponsor on 3 April 2012.

The experimental start date was 11 March 2012 and the experimental completion date was 

23 March 2012.
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2. Test substance

Identification: 2-Bromo-3,3,3-trifluoropropene

CAS Number: 1514-82-5

Action: Fire extinguishing agent

Description: Clear liquid with slight yellow tint

Storage conditions: Refrigerator (nominally 4C)

Batch number: KgF080

Date of receipt: 12 September 2011

Expiry date: 1 August 2012

Purity: 99.9% excluding stabilisers

Stabilisers:
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3. Experimental Procedure

3.1 Animal management

Animals for this study were selected from a stock supply of healthy adult rabbits of the New 

Zealand White strain. They were in the weight range of 3.55 to 4.94 kg and 35 or 36 weeks 

of age, prior to treatment (Day 1). All rabbits were acclimatised to the experimental 

environment for a period of ten weeks prior to the start of the study.

The rabbits were housed individually in plastic cages with perforated floors. Each rabbit was 

offered 125 g of a standard laboratory rabbit diet per day; drinking water was provided ad 

libitum. The batch of diet used for the study was analysed for nutrients, possible

contaminants and micro-organisms likely to be present in the diet and which, if in excess of 

specified amounts, might have an undesirable effect on the test system. The animals were 

given a dietary supplement of hay.

During the acclimatisation and study period the animals were given small soft white 

untreated wood blocks for environmental enrichment.

Results of routine physical and chemical examination of drinking water, as conducted by the 

supplier are made available to Huntingdon Life Sciences Limited.

Animal room environmental controls were set to maintain temperature within the range 16 to 

20°C, and relative humidity within 40 to 70%. These environmental parameters were 

recorded and the permanent record archived with other departmental raw data. Lighting was 

controlled by means of a time switch to give 12 hours of artificial light (06:00 to 18:00 GMT) 

in each 24 hour period.

Each animal was identified by a numbered tag placed through the edge of one ear. This 

identification was unique within the Department throughout the duration of the study. Each 

cage was identified by a coloured label displaying the study number and animal number.

3.2 Test substance preparation

2-Bromo-3,3,3-trifluoropropene was administered as supplied by the Sponsor.

The absorption of 2-Bromo-3,3,3-trifluoropropene was not determined.

The identity, strength and purity of the test substance received, its stability under the storage 

conditions and the conditions of administration were the responsibility of the Sponsor.
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3.3 Treatment procedure

On the day before application of the test substance, hair was removed with clippers from the 

dorso-lumbar region of each rabbit exposing an appropriate sized area of skin.

Approximately 0.5 ml of the test substance was applied under a 2-ply 25 mm x 25 mm porous 

gauze pad secured with ‘blenderm’ surgical tape to intact skin sites on three animals. An 

additional site was similarly treated with the exception of test substance and acted as a 

control. Each treatment site was covered with a square piece of aluminium foil 

(approximately 55 mm x 55 mm) all four sides of which were secured to the skin by 

impermeable surgical tape (Blenderm).

A single animal (number 5) received three exposures of three minutes, one or four hours 

duration in a step-wise manner and acted as a preliminary screen. In the absence of a severe

effect on removal of the dressings the next exposure was initiated. In the absence of a severe 

response in this animal, two further animals were committed to the study; each animal 

received a single four hour exposure.

For exposures of one hour or more each treatment site was covered with cotton wool and 

"Tubigrip" elasticated bandage dressing for the duration of the exposure period. The animals 

were returned to their cages immediately after treatment.

At the end of the exposure period the semi-occlusive dressing and gauze pad were removed 

and the treatment site was washed with lukewarm water (30-40°C) to remove any residual 

test substance. The treated area was blotted dry with absorbent paper.

3.4 Serial observations

3.4.1 Clinical signs

All animals were observed daily for signs of ill health or toxicity.

3.4.2 Dermal responses

Examination of the treated skin was made on removal of the dressings (for 3 minute or one 

hour exposures) and approximately 1, 24, 48 and 72 hours later. Only the data for the four 

hour exposure are reported, the data from the three minute and one hour exposures are held in 

the archives. 

Local dermal irritation was assessed using the prescribed numerical system:

Erythema and eschar formation:

No erythema 0

Very slight erythema (barely perceptible) 1

Well-defined erythema 2

Moderate to severe erythema 3

Severe erythema (beet redness) or eschar formation (injuries in 

depth) preventing grading of erythema

4
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Oedema formation:

No oedema 0

Very slight oedema (barely perceptible) 1

Slight oedema (edges of area well-defined by definite raising) 2

Moderate oedema (raised approximately 1 millimetre) 3

Severe oedema (raised more than 1 millimetre and extending 

beyond the area of exposure)

4

3.4.3 Interpretation of responses

Primary Irritation Index

A primary irritation index (PII) was calculated from the erythema and oedema scores 

according to the following formula as described in Technical Report No. 66 “Skin irritation 

and Corrosion: Reference chemicals data bank” (March 1995) ECETOC, Brussels.

PII=  Erythema at 24/48/72 hours +  Oedema at 24/48/72 hours

3 x number of animals

The maximum possible score was 8.0. The PII was then used to classify the test substance as 

follows:

Primary Irritation Index Classification

0 non-irritant

>0 - 2.0 mildly irritating

2.1 - 5.0 moderate irritant

5.1 - 6.0 moderate to severe irritant

> 6.0 severe irritant

This classification system is a modification based on “Appraisal of the Safety of Chemicals in 

Foods Drugs and Cosmetics” published by the Association of Food and Drug Officials of the 

United States, 1959.
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Globally Harmonised System (United Nations) 

European Commission regulation 1272/2008 contains the following criteria for classification 

of irritants and corrosives to skin. These were derived from the Globally Harmonised System 

(United Nations 2005 and more recent revisions). 

Corrosion: a corrosive substance is a substance that produces destruction of skin tissue, 

namely, visible necrosis through the epidermis and into the dermis, in at least one tested 

animal after exposure up to four hours duration. Corrosive reactions are typified by ulcers, 

bleeding, bloody scabs and, by the end of observation at 14 days, by discolouration due to 

blanching of the skin, complete areas of alopecia and scars. Corrosive substances are 

assigned to Category 1; three subcategories are provided within the corrosive category: 

Subcategory 1A: where responses are noted following up to 3 minutes 

exposure and up to 1 hour observation 

Subcategory 1B: where responses are described following exposure 

between 3 minutes and 1 hour and observations up to 14 days 

Subcategory 1C: where responses occur after exposures between 

1 hour and 4 hours and observations up to 14 days 

Irritation: an irritant substance is a substance that produces reversible damage to the skin 

following an exposure of up to four hours. Irritant substances are assigned to Category 2; the 

following thresholds apply: 

Mean value of >2.3 - <4.0 for erythema/eschar or for oedema in at 

least 2 of 3 tested animals from gradings at 24, 48 and 72 hours after 

patch removal or, if reactions are delayed, from grades on 3 

consecutive days after the onset of skin reactions. 

Inflammation that persists to the end of the observation period 

normally 14 days in at least 2 animals, particularly taking in to 

account alopecia (limited area), hyperkeratosis, hyperplasia, and 

scaling. 

In some cases where there is pronounced variability of response 

among animals, with very definite positive effects related to chemical 

exposure in a single animal but less than the criteria above. 

3.4.4 Termination 

Following completion of the observation period the animals were humanely killed by an 

intravenous injection of sodium pentobarbital. 
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3.5 Archives

All raw data arising from the performance of this study at Huntingdon Life Sciences is the 

property of the Sponsor and will be lodged together with a copy of the final report in the 

Huntingdon Life Sciences Archive.

Such records will be retained for a minimum of one year from the date on which the Study

Director signs the final report. At the end of the retention period the Sponsor will be 

contacted and advice sought on the return, disposal or further retention of the records.

Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study and 

a copy of the final report in its archive indefinitely.

3.6 Deviations from protocol

The study protocol indicated that the test substance should be stored refrigerated 

(nominally 4C), desiccated and under a nitrogen blanket, however, the material was stored 

refrigerated without neither desiccant nor nitrogen blanket.

The Sponsor advised that to evaluate the material, a couple of tests should be performed:

1. Appearance: the material should be a clear liquid with a slight yellow colour and no 

particulates. 

2. pH: the pH of an aqueous 50% v/v preparation should be determined. Shake a 1:1 

mixture of the test material and distilled water for 30 seconds, then, allow the two 

layers to separate (about 2 minutes) and using a calibrated pH meter measure the pH 

of the aqueous layer. 

If there is no change in the appearance and the pH value is above 5 then the material would 

be considered to have been unaffected.

The appearance was considered to have been unchanged and the pH value for the working 

container for this study was found to be 6.60. Accordingly, it is considered that the incorrect 

storage conditions did not affect the integrity of the test substance.

There was no other deviation from protocol. 
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4. Results

4.1 Clinical signs

There was no sign of toxicity or ill health in any rabbit during the observation period.

4.2 Dermal responses

No dermal reaction was observed in any animal throughout the duration of the study.
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5. Conclusion

According to the criteria of the Globally Harmonised System and European Commission 

regulation 1272/2008 2-Bromo-3,3,3-trifluoropropene did not produce irritation or corrosivity 

and did not require any classification and labelling.
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Table 1 Mean values for erythema and oedema

24, 48 and 72 hours after removal of dressings *

Animal number 

and sex

Erythema Oedema

Test Control Test Control

5 F 0.0 0.0 0.0 0.0

78 F 0.0 0.0 0.0 0.0

79 F 0.0 0.0 0.0 0.0

* 4 hour exposure

F Female
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Table 2 Individual values for erythema and oedema

Test site: 0.5 mL 2-Bromo-3,3,3-trifluoropropene } semi occluded for four hours

Control site: No treatment

Animal number 

and sex

Type of 

Response

Score after removal of dressings

1 hour 24 hours 48 hours 72 hours

Test site Control site Test site Control site Test site Control site Test site Control site

Erythema 0 0 0 0 0 0 0 0

5 F #

Oedema 0 0 0 0 0 0 0 0

Erythema 0 0 0 0 0 0 0 0

78 F

Oedema 0 0 0 0 0 0 0 0

Erythema 0 0 0 0 0 0 0 0

79 F

Oedema 0 0 0 0 0 0 0 0

F Female

# Sentinel animal
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Annex 1 Weight of evidence 

Prior to undertaking in-vivo irritation testing the Study Director conducted a weight-of-theevidence analysis to ensure that the in-vivo testing was sufficiently justified. The following 

factors have been taken into consideration: 

1. Nature of test substance. 

A clear liquid with a slight yellow tint for use as a fire extinguishing agent. The sponsor 

advises the following: 

Chemical name: 2-Bromo-3,3,3-trifluoro-1-propene 

Molecular weight: 174.95 

Purity: 99.9% excluding stabilisers 

Boiling point: 34°C 

2. Evaluation of existing human and animal data (including dermal toxicity) 

Internet searches indicated that the material is extremely flammable and harmful or an 

irritant. These indications were clarified with “the toxicological properties of this product 

have not been fully determined” and are considered to be precautionary. 

3. Analysis of structure activity relationships (SAR). 

None available. 

4. Physicochemical properties. 

Physicochemical properties (HLS study WAG0005) indicated that the test material is not 

flammable and the partition coefficient is 2.7. 

pH recorded at HLS as a 10% aqueous preparation: 5.2 

5. Results from in-vitro or ex-vivo tests. 

Due to the low evaporation point and the small amount of test material used in the EpiSkin 

assay, the test material was considered unsuitable for testing by this method. 

The Physicochemical properties show a lack of autoflammability and flash point, however, 

data available on the InterNet quotes a flash point in the region of -23°C to -25°C. These 

MSDS’s also state that the “chemical, physical and toxicological properties have not been 

thoroughly investigated” so it is considered that the extremely flammable and -25°C flash 

point labels are precautionary based on its volatility; any irritation label is likewise 

considered to be precautionary. 

It is generally accepted that compounds with a partition coefficient in the range of 1 to 4 

(optimally between 1 and 2) and a molecular weight of less than 500 may be absorbed, at 

least in part, via the dermal route. The partition coefficient of 2-Bromo-3,3,3-trifluoro-1-

propene is 2.7 and the molecular weight is 174.95; accordingly, it was considered likely that 

this material will cross the skin barrier. The low boiling point (below body temperature) 

indicates that the material will not be in contact with the body for a long period of time, 

although steps (airtight administration) were taken to maximise exposure time. 

The Study Director was satisfied that, in the absence of obtainable data, there was sufficient 

justification to conduct an in-vivo rabbit skin irritation study. 
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Annex 2 GLP Compliance Statement